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Time and Temperature

Antibody concentration, temperature, and time interact in the staining reaction: higher antibody concentrations reach effective staining saturation more quickly than lower concentrations and any concentration reaches saturation faster at a higher temperture. Even though azide is a metabolic poison (and some labs find that it allows them to stain cells at room temperature, rapidly and with impunity), most labs stain with all reagents/solutions at 4°C even in the presence of azide to be sure of minimizing capping / internalization/ miscellaneous loss of surface-bound antibodies. At 4°C, 30 minutes is a reasonable staining time. Antibody titrations can all be done at 4°C and for 30 minutes. After titration to determine a saturating antibody concentration, check out the effect of time and use a time that is long enough that a bit more time or a bit less time makes no difference to the results.

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